7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation.
OBJECTIVE
7-Ketocholesterol is a highly toxic oxysterol found in abundance in atherosclerotic plaques and is believed to play a critical role in atherosclerosis. The purpose of this study was to identify and localize 7-ketocholesterol (7kCh) in the primate retina and to examine the potential consequences of its presence in oxidized lipid deposits in the retina.


METHODS
Unsterified 7kCh was identified and quantified by high-performance liquid chromatography-mass spectrometry. Localization of 7kCh was performed by immunohistochemistry. VEGF induction was determined by qRT-PCR. Cell viability was determined by measuring cellular dehydrogenase activity. Analyses were performed using ARPE19 and human vascular endothelial cells (HMVECs).


RESULTS
7-Ketocholesterol is localized mainly to deposits in the choriocapillaris and Bruch's membrane and on the surfaces of vascular endothelial cells of the neural retina. RPE/choriocapillaris regions contained approximately four times more 7kCh than the neural retina. In ARPE19 cells and HMVECs, oxidized LDL and 7kCh induced VEGF 8- to 10-fold above controls. Hypoxia inducible factor (HIF)-1alpha levels did not increase as a result of 7kCh treatment, suggesting an HIF-independent induction pathway. Cholesterol sulfate, a liver X receptor (LXR) antagonist, had marked attenuation of the 7kCh-mediated VEGF induction. LXR-specific siRNAs also reduced VEGF induction. Inhibition of NF-kappaB with BAY 11-7082 reduced IL-8 but not VEGF induction.


CONCLUSIONS
The location of 7-kCh in the retina and its induction of VEGF in cultured RPE cells and HMVECs suggest it may play a critical role in choroidal neovascularization. The pathway for VEGF induction seems to be independent of HIF-1alpha and NF-kappaB but seems to be partially regulated by LXRs.
Moreira
Ernesto F
EF
Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Larrayoz
Ignacio M
IM
Lee
Jung Wha
JW
Rodríguez
Ignacio R
IR
D000818:Animals; D016570:Bruch Membrane / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D002788:Cholesterol Esters / Q000378:metabolism; D020256:Choroidal Neovascularization / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007653:Ketocholesterols / Q000378:metabolism* / Q000494:pharmacology; D050356:Lipid Metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology; D000071518:Liver X Receptors; D008253:Macaca mulatta; D013058:Mass Spectrometry; D016328:NF-kappa B / Q000378:metabolism; D057093:Orphan Nuclear Receptors; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D002788:Cholesterol Esters; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007653:Ketocholesterols; D008077:Lipoproteins, LDL; D000071518:Liver X Receptors; D016328:NF-kappa B; D057093:Orphan Nuclear Receptors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C099252:oxidized low density lipoprotein; C007045:cholesteryl sulfate; C003001:7-ketocholesterol